The Effect Of Gut Microbiota On Axonal Growth And Functional Recovery After Intracerebral Hemorrhage In Mice And Its Related Mechanism

Objective: Intracerebral hemorrhage refers to the non-traumatic rupture of the cerebrovascular caused by hypertension,cerebral arteriosclerosis and other causes.Intracerebral hemorrhage is one of the most common subtypes of stroke.The annual incidence of intracerebral hemorrhage disease accounts for about 10%-15% of the total incidence of stroke.Intracerebral hemorrhage can lead to a series of rapid pathological changes in the brain,including hematoma compression effect,brain tissue edema,inflammatory response and neuronal cell apoptosis.The rapid deterioration of early symptoms leads to an extremely high rate of disability and mortality of intracerebral hemorrhage,and the patient's neurological function is seriously damaged.At present,the clinical treatments of intracerebral hemorrhage mainly include absolute bed rest,reducing intracranial pressure and surgery,which are only applicable to the acute stage.It is worth noting that the self-repair and plasticity of the brain may provide a relatively appropriate time-window for the treatments of intracerebral hemorrhage in the recovery period.Brain injury can stimulate the function of brain nerve self-repair,and its main mechanisms include axon regeneration,nerve excitability changes,neurogenesis and angiogenesis.Microbial-Enteric-Brain axis is a bidirectional interaction pathway between intestinal microbiome and central nervous system,including immune,endocrine,metabolic and nervous pathways.The occurrence of central nervous system diseases can lead to the disbiosys of gut microbiota;oppositely,remodeling homeostasis can also alleviate the symptom and progression of central nervous system diseases.Dietary fiber is essentially a kind of polysaccharide,which was once considered as a "nonnutritive substance" because it could not be directly absorbed and generated energy by the gastrointestinal tract.However,with the in-depth study of nutrition and related sciences,it has been found that dietary fiber can be metabolized and utilized by gut microbiome and produce Short-chain fatty acid(SCFA).Short-chain fatty acids are organic fatty acids that can be rapidly absorbed by the intestine.They play an important role in maintaining the normal function of the intestine,regulating the permeability of the blood-brain barrier,maintaining the stability of the internal environment of the central nervous system,and affecting the development of the brain and neurological function.Antibiotics are secondary metabolites produced by microbiome or higher animals and plants in the course of life with antipathogen effect,which can significantly change the type and quantity of gut microbiota and induce changes in the microecology and peripheral immune response of gut microbiota.However,it is still unclear whether regulating gut microbiota can affect axonal growth and neurological function recovery after intracerebral hemorrhage from the early stage to the chronic stage.Therefore,the purpose of our study is exploring the effects of gut microbiota change on axon growth and neurological function recovery after intracerebral hemorrhage in mice and its related mechanisms.Methods: In our study,7-week-old male C57BL/6 mice with a body weight of 20?25g were provided by Beijing Weitong Lihua Biotechnology Co.,Ltd.,and these mice were randomly divided into SHAM operation group(SHAM group),hemorrhage group(ICH group),hemorrhage+high fiber diet group(ICH+HFD group),and hemorrhage+antibiotics group(ICH+ AB group).Focal intracerebral hemorrhage was induced by injection of Collagenase VII into the right striatum of the brain in a stereotactic manner according to the anatomical atlas of the mouse brain,and the mouse cerebral hemorrhage model was established.On the 28 th day after intracerebral hemorrhage,biotinylated dextran amine(BDA)was injected into the motor cortex of the contralesional side of the brain in a stereotactic manner to mark the corticospinal tract anterograde.From day 1 to35 after ICH,high fiber diet was added to ICH+HFD group and antibiotic water was added to ICH+AB group,respectively.On the 35 th day after intracerebral hemorrhage,balance beam-walking test and cylinder test were used to evaluate the motor function of the affected limbs of mice.Mice in each group were killed at day 35 after intracerebral hemorrhage.According to the purpose of the experiment,mice needing cardiac perfusion were fully anesthetised with isoflurane and perfused with 0.9% normal saline and 4%paraformaldehyde.The brain tissue and cervical spinal cord tissue of mice were extracted completely after their limbs were completely stiff.After fixation,dehydration and embedding,the frozen sections of brain(10?m thick)and spinal cord(30?m thick)were prepared by a cryosicicle machine,and then immunofluorescence staining was performed.Mice requiring direct freeze-storage samples were rapidly brain extracted after cardiac perfusion with normal saline,washing the residual blood on the surface and stored in a refrigerator at-80?.We used a confocal microscope to photograph multiple coherent layers in the Z-axis direction of mouse spinal cord sections.Then,the axon fibers were reconstructed in 3D.NIH Image J software was used to trace and measure the length of new axons,so as to observe the sprouting of BDA-labeled corticospinal tract fibers.On the35 th day after intracerebral hemorrhage,we used Western blot to examine the expression levels of phosphor-AKT,phosphor-NF?B,PSD-95,BDNF,Nogo A,GAP43,Synaptophysin and other indexes in the iplesional cortex of mice.Statistical analysis software SPSS 26.0 for Windows was used for all data analysis.The experimental data were expressed as mean ± standard deviation.One-way ANOVA and LSD-T test were used to determine the statistical significance of the differences,P < 0.05 has statistical difference.Results:(1)Effects of intracerebral hemorrhage,HFD and antibiotics on gut microbiota:Firmicutes in the ICH group became the dominant bacteria compared with the SHAM group;Compared with ICH group,the number of Bacteroidetes in ICH+HFD group was significantly increased,while the number of Firmicutes was unchanged.Compared with ICH group,the number of Firmicutes was unchanged,but Proteobacteria was significantly increased in ICH+AB group.(2)Effects of intracerebral hemorrhage,HFD and antibiotics on axon growth: Compared with Sham group,the length of axons in ICH group was significantly increased(P < 0.001);The axon length of ICH+HFD group and ICH+AB group was further increased than ICH group(P < 0.05).(3)There was no significant difference in behavioral function among all groups(P > 0.05).Conclusion:(1)Significant changes in gut microbiota occurred after intracerebral hemorrhage.(2)HFD and antibiotics interventions significantly changed the composition of gut microbiota after intracerebral hemorrhage.(3)HFD and antibiotics interventions have an increased effect on axonal growth in chronic intracerebral hemorrhage.(4)The effects of HFD and antibiotics interventions on motor function recovery in chronic period after intracerebral hemorrhage were not statistically significant.